Recent evidence indicates that the most powerful anti-tumor T cells recognize neoantigens derived from unique mutated proteins expressed by an individual tumor, suggesting that precision vaccination is required to induce effective anti-tumor immunity in cancer patients. Importantly, such anti-tumor T cells cause tumor regression at advanced disease stages upon therapeutic immune checkpoints blockade. We have pioneered studies exploring the use of local tumor radiotherapy (RT) as a means to generate an in situ individualized vaccine. We were the first to demonstrate in a pre-clinical model that RT sensitizes unresponsive tumors to CTLA-4 blockade by inducing T cells specific for endogenous tumor antigens. Emerging evidence by us and others suggests a similar effect of RT in the clinic. However, lack of knowledge about the mechanisms involved precludes rapid progress towards the effective use of RT as an immune adjuvant. One critical unanswered question is whether dose and fractionation affect RT ability to elicit anti-tumor immune responses. We have previously found in two mouse carcinomas that generation of an in situ vaccine synergistic with anti-CTLA-4 treatment in inducing immune-mediated regression of irradiated and synchronous non-irradiated tumors (abscopal effect) was achieved by RT given in 3 fractions of 8 Gy but not by a single 20 Gy dose, suggesting that the RT regimen employed is critical. We now have data supporting the hypothesis that carcinoma cell-intrinsic activation of type I interferon (IFN-I) pathway by RT is required to generate an in situ vaccine, suggesting that RT triggers canonical defense pathways in neoplastic epithelial cells that mimic a viral infection. Our data also indicat that fractionated (FRT) but not single dose (SDRT) radiation can accomplish this via activation of the cytosolic DNA sensor cyclic GMP-AMP synthase (cGAS) and downstream stimulator of IFN genes (STING) within the irradiated cancer cells. To test the above hypothesis several mouse and human carcinoma cells will be employed to determine which RT dose/fractionation activates cGAS/STING. Next, in vivo experiments using cancer cells with selective knockdown of cGAS or STING implanted in syngeneic immunocompetent wild type, cGAS-deficient, and STING-deficient mice will be performed to determine the role of this pathway in RT-mediated induction of anti-tumor T cells that mediate tumor regression and abscopal responses. To determine whether the effects of IFN-I produced by irradiated cancer cells are cancer cell autonomous or require signaling in host DC, IFNAR1-deficient mice will be used as tumor recipient. The relationship between cancer cell-derived IFN-I and DC recruitment to tumors will be established. Finally, we will investigate the mechanisms whereby RT activates the cGAS/STING pathway in cancer cells. Overall, data obtained will have important implications for a novel use of radiotherapy as a relatively simple and widely available modality for individualized tumor vaccination.